Method of structure-based drug design using a crystalline form of activated trap

ABSTRACT

A crystalline form of mammalian TRAP (tartrate-resistant and purple acid phosphatase) is described. The enzyme is activated by cleavage prior to crystallization with a protease and the crystalline form of the mammalian TRAP is capable of being used for X-ray studies.

The present application is a divisional of U.S. application Ser. No.09/451,900, filed Dec. 1, 1999, which issued as U.S. Pat. No. 6,329,184,on Dec. 11, 2001, which claims priority to U.S. Provisional ApplicationSer. No. 60/113,304, filed Dec. 22, 1998, and Swedish Patent ApplicationNo. 9804418-3, filed Dec. 18, 1998. These applications are incorporatedherein by reference in their entirety.

The invention relates to crystalline form of TRAP (tartrate-resistantand purple acid phosphatases), which is activated by cleavage. The TRAPis preferably human or rat TRAP. The atomic structural coordinates aregiven as well as the conserved surface which is created by specialatoms, relevant for the design of modulators or inhibitors of the humanTRAP. This crystalline form of activated TRAP can be used forstructure-based drug design for specific modulator, activator orinhibitor of TRAP activity, useful in the treatment of diseases ordegenerative conditions resulting in increased bone resorption, such astissue damages, bone metabolic disorders, osteoporosis.

BACKGROUND

Tartrate resistant purple acid phosphatase (TRAP or PAP) is a mammaliandi-iron containing enzyme highly expressed in a limited number oftissues. In humans and rodents it is primarily present in cellsresponsible for bone resorption, osteoclasts, and in macrophages ofspleen, liver and lung.

Normal bone function requires a turnover of bone. Bone is constantlybeing rebuilt by cycles of resorption and formation which means thatformation is closely linked to resorption (a phenomenon referred to ascoupling).

TRAP is an enzyme expressed predominantly in bone resorbing cells(osteoclasts). Investigations in TRAP knockout mice show that theresorption process is disrupted so that, with increasing age, TRAPknockout mice become osteopetrotic, i.e. have an increased bone mineralcontent and more dense bone is formed. Osteoclasts prepared from theseanimals are functional and do resorb bone but to a lesser extent thanwild type mouse osteoclasts.

Phosphatases are enzymes that remove organic phosphates from proteins.The mammalian Purple Acid Phosphatases (PAPs), a group of enzymes towhich Tartrate Resistant and purple Acid Phosphatase (TRAP) belongs, arecharacterized by a binuclear iron center at the active site.

The binuclear iron center, low pH optimum (≈5), high isoelectric point(≈9) and insensitivity to inhibition by L(+) tartrate are features ofTRAP that may be involved in the apparent substrate specificity at thelow pH in the osteoclastic resorption area. The TRAP enzyme is acationic glycoprotein with a molecular mass of 35 kD. The rat TRAP is aprotein with a monomeric 306 amino acid peptide structure. See FIG. 1.The peptide sequence of rat bone TRAP displays 89-94% homology to TRAPenzyme of the human placenta, bovine spleen, and uteroferrin.

TRAP hydrolyzes aryl phosphates, nucleoside di- and triphosphates,pyrophosphate and phosphoproteins. Its physiological role remainsunclear but TRAP may mediate dephosphorylation of bone matrix proteinssuch as osteopontin and bone sialoprotein. Dephosphorylation of bonematrix proteins enables osteoclasts to migrate over the bone surface andTRAP is therefore likely to be involved in the attachment of osteoclaststo the bone surface.

In humans and rats, PAP enzymes are highly expressed in certain cells ofthe monocytemacrophage lineage, such as the bone-resorbing osteoclastsand certain activated macrophages in spleen, liver and lung [1-4], andTRAP has since long been used as a histochemical marker for these cells.Given the broad substrate specificity of PAP enzymes, it is conceivablethat other factors, such as local availability and propercompartmentalisation of PAPs with their potential substrates, are otherimportant factors in determining the physiological action of PAPs inbiological systems.

The cDNA sequences of TRAP/PAP enzymes from different species and organsall indicate that these enzymes are translated as a single polypeptideof around 35 kDa [5-8]. This contrasts with the predominantly twosubunit structure, consisting of a 20-23 kDa N-terminal domain linkedthrough a disulphide bond to a 15-17 kDa C-terminal domain, observed inpurified enzyme preparations from a variety of sources including humanand rat bone [9-10], giant cell tumors [11] and normal and pathologicalspleen [12-14]. In contrast, uteroferrin purified from endometrialsecretions are mostly in the single subunit form [12, 15] as are therecombinant PAPs generated by overexpression using the Baculovirussystem [16, 17, 18]. Orlando et al [13] managed to separate themonomeric and two-subunit variants of PAP from bovine spleen, anddemonstrated a markedly higher specific enzyme activity associated withthe two subunit form. Moreover, digestion of the single subunit formwith the serine proteases trypsin or chymotrypsin generated the 23 kDaand 15 kDa disulfide-linked fragments characteristic of the two subunitform together with a significant enhancement of enzyme activity. Similarnicking and activation of the non-cleaved purified recombinant human andmouse PAPs were noted upon prolonged storage [17].

Purple acid phosphatases (PAPs) are acid metallohydrolases that containa binuclear Fe3+M2+ center in their active site, where M=Fe or Zn[19-22]. In mammals, these enzymes are also referred to astartrate-resistant acid phosphatases (TRAPs) (EC 3.1.3.2) or type 5 acidphosphatases[23]. TRAPs are iron-containing, monomeric glycoproteinswith molecular weights of around 35,000 Da [24]. The deduced amino acidsequences of human, rat and mouse TRAPs shows a high degree of identityto the mammalian members of the PAP family, e.g uteroferrin (Uf) andbovine spleen PAP[5-7]. Recently, EPR spectroscopic analysis of ratrecombinant TRAPs[16] have provided compelling evidence that this enzymebelong to the purple acid phosphatase family.

Mammalian PAPs contain a FeFe centre, while a plant PAP from red kidneybeans (KBPAP) instead has a FeZn center [25]. The anti-ferromagneticallyspin-coupled binuclear iron centre of the mammalian PAPs exists in twostable interconvertible states: pink, reduced, EPR-visible andenzymatically active, with a mixed-valent Fe2+-Fe3+ cluster; and purple,oxidized, EPR-silent and catalytically inactive, with the binuclear pairas Fe3+-Fe3+ [21, 26-27]. In contrast, the plant enzyme with amixed-valent Zn2+-Fe3+ centre is constitutively active [28 ]. The M(2+)site in the PAPs can harbour either Zn2+ or Fe2+ without alteration ofenzyme activity or spectral properties [28-30]. KBPAP is the only PAPwhose X-ray structure has been determined. [17] The active site of KBPAPconsists of an iron and a zinc ion bridged by an aspartate and probablya hydroxide. The Fe3+ site is coordinated by tyrosine, histidine andaspartate, while the Zn2+ site is coordinated by two histidines and anasparagine [17,31]. One solvent molecule is probably bound to each metalion. Kidney bean PAP is a homodimeric protein with a molecular weight ofaround 110,000 Da, and exhibits a low overall sequence homology to themammalian PAPs [32]. However, an alignment of the sequences of Uf andKBPAP displays an identical positioning of the amino acid residuesligating the di-metal centre [31,32]. Moreover, the mammalian proteinphosphatases calcineurin (type 2B) [1-2] and protein phosphatase type 1(PP-1) [3-4] both contain a di-nuclear metal centre and also reveal astriking similarity to the plant PAP enzyme in the coordinationenvironments of the active site, except for the absence of the tyrosineligand. These two latter enzymes are serine/threonine proteinphosphatases, suggesting that also PAPs function as proteinphosphatases. A sequence motif, DXH(X)nGDXXD(X)nGNHD/E, incorporatingmost of the metal-coordinating amino acids found in the PAP and PPstructures so far identified has recently been identified also in alarge group of phosphoesterases, including other phosphomonoesterases,nucleotide phosphatases and nucleases, from plants, bacteria and animalcells [8-11]. This phosphoesterase signature motif is represented at thesecondary structure level as a β-α-β-α-β-fold that serves to positionthe two metal ions at the active site with four of the metal ligandsprovided by loop residues between each β-sheet and α-helix. Theimportance of this motif has been confirmed by site-directed mutagenesisstudies [12-13]. Furthermore, the PAP members are related to asuperfamily of μ-(hydr)oxo-bridged binuclear iron proteins, includinghemerythrin, R2-subunit of ribonucleotide reductase, methanemonooxygenase hydroxylase and others [15]. All members of thissuperfamily of iron-oxygen proteins contain a binuclear iron center buthave different functions.

No crystallisation of TRAP, nor of actived TRAP has earlier beenperformed. The crystal form of the new active form of TRAP is of greatuse in the screening for specific modulators, activators or inhibitor ofTRAP activity. Such specific modulators, activators or inhibitor areuseful in the treatment of diseases or degenerative conditions resultingin increased bone resorption, such as tissue damages, bone metabolicdisorders, osteoporosis.

FIGURES

FIG. 1. The amino acid sequence of rat TRAP, Sequence listing No 1.

FIG. 2. A view of TRAP.

FIG. 3. Secondary structure diagram of the catalytic domains of TRAP

FIG. 4. A tracing of α-carbons in TRAP.

FIG. 5. A view of the active site of TRAP.

FIG. 6. SDS-PAGE gel showing the contents of the crystal containingdrops.

THE INVENTION

The present invention relates to crystalline form of TRAP(tartrate-resistant and purple acid phosphatases), which is activated bycleavage, preferably human or rat TRAP (Sequence listing No 1). Theatomic structural coordinates are given in Table 2. The conservedsurface which is created by special atoms is important and crucial forthe design of modulators or inhibitors of the human TRAP.

This crystalline form of activated TRAP can be used for structure-baseddrug design for specific modulator, activator or inhibitor of TRAPactivity, useful in the treatment of diseases or degenerative conditionsresulting in increased bone resorption, such as tissue damages, bonemetabolic disorders, osteoporosis.

This claimed crystal form of TRAP is a cleaved form. The cleavage ofTRAP increases activity and gives the TRAP, which here is calledactivated TRAP. Reference is given to U.S. patent application Ser. No.09/442,816.

The invention is defined in the attached claims.

EXAMPLE Methods

Protein Production and Purification

Purification of baculovirus produced recombinant TRAP was performed asdescribed [16].

Crystallisation

The protein was crystallized by vapor diffusion with the hanging dropmethod. The crystals used in the structure determination were grown witha reservoir containing 16% PEG 8000, 0.1 M HEPES(buffer+precipitant+salt) pH 7.0 and 0.1 M KH₂PO₄. The drop consisted of2 μl protein solution and 2 μl from the reservoir. The proteinconcentration was 5-8 mg/ml. Purple crystals appeared within two weeksat 18 C and reached a maximum size of 0.1×0.1×0.05 mm.

Using different conditions, different crystal forms were obtained butnot all of these crystals were suitable for X-ray studies. Common to allsuccessful crystallisation attempts was the presence of inorganicphosphate.

Data Collection

Most X-ray data was collected on an inhouse Raxis-4 imageplate detectormounted on a Rigaku RU300 rotating anode. A two-wavelength MAD datasetwas also collected on beamline BL14 at ESRF, Grenoble. All datacollection was performed under cryoconditions. Prior to freezing, thecrystal was transferred for a few seconds to a cryosolution containingtwo parts reservoir solution and one part glycerol. The space group was4₁, with cell dimensions a=b=116.4 Å and c=63.3 Å. The best crystaldiffracted to 2.7 Å resolution.

Three distinct heavy atom derivative crystals were produced, usingHg(CN)₂/HgCl₂,K₂PtCl₄ and Na₂WO₄. Heavy atom derivatives were preparedby adding small volumes (approximately 0.1 μl) of saturated heavy atomsolutions to the crystal containing drop two hours prior to freezing.The mercury solution contained equal amounts of Hg(CN)₂ and HgCl₂.

The diffraction images were processed with DENZO and Scalepack [33],conserving the anomalous differences for all datasets, including thenative. Native and derivative datasets were put to a common scale withthe program Scaleit.

Structure Determination and Refinement

The strongest heavy atom site for the mercury derivative was found bydifference Patterson map analysis. The other sites were located usingdifference Fourier syntheses. Mlphare [34] was used for heavy atomparameter refinement and phasing at this stage. Anomalous differenceFouriers were calculated to confirm heavy atom sites and identify thecorrect enantiomer. The phases were improved by solvent flattening in dm[35] and the corresponding electron density map to 3.5 Å resolution wasused for most of the structure interpretation. Later on the programsSharp [36] and Solomon [37] were used to produce an electron density mapof superior quality to 2.7 Å. A partial model of kidney bean acidphosphatase was positioned into the density to facilitate modelbuilding. The two highest peaks in the anomalous difference Fourier ofthe native data corresponded well with the positions of the two metalbinding sites in the positioned model of the kidney bean enzyme. A modelof hTRAP was built with O [38] with intermittent rounds of refinement inCNS [39]. The progress of model improvement was monitored with Procheck[40]. The model consists of all residues for human TRAP, except amissing loop-region (residues 146-160) and the last four residues. TheR-factor is 23% and the free R is 31% for reflections in the interval50-2.7 Å. 82% of the residues fall in the most favorable regions of theRamachandran plot as defined by Procheck. Data collection and refinementstatistics are summarized in Table 1. The crystal structure giving theatomic structural coordinates is given in Table 2.

TABLE 1 Crystallographic data Data collection resolution λ Completeness(%) Nr of Phasing Data set (Å) (Å) (redundancy) Rsym^(a) Riso^(b) sitesPower^(c) native 2.7 1.542 89(3.9) 0.107 — — — K₂PtCl₄ 3.0 1.542 69(2.2)0.065 0.433 6 1.4 Na₂WO₄ 6.0 1.542 93(2.2) 0.101 0.288 3 1.5 mercury-13.0 1.542 88(3.7) 0.076 0.363 4 2.1 mercury-2 4.0 1.008 72(2.6) 0.0590.290 4 3.2 mercury-3 4.0 0.918 68(2.7) 0.080 0.279 4 2.8 RefinementR-factor: 0.23; Free R: 0.31 ^(d)Ramachandran angle distribution (%):most favourable: 78.9, allowed: 18.6, generously allowed: 1.2,disallowed: 1.2 ^(a)R_(sym) =Σ_(h)Σ_(i)|I(h)−<I(h)_(i)>|/Σ_(h)Σ_(i)I(h)_(i), where <I(h)> is theaverage intensity of reflection h, Σ_(h) is the sum over allreflections. and Σ_(i) is the sum of all measurements of reflection h.^(b)R_(iso) = Σ_(h)|F_(PH) − F_(P)|/Σ_(h) F_(P), where F_(PH) and F_(P)are the derivative and native structure factor amplitudes. ^(c)Phasingpower = F_(h)/lack of closure. Acentric reflections. ^(d)Values anddefinitions from Procheck (49). mercury-1,2,3: mixture of saturatedsolutions of Hg(CN)₂ and HgCl₂.

The Structure

The structure of TRAP can be described as a double beta sheet sandwhichsurrounded on both sides by alpha helices and has the overall foldobserved for the C-terminal domain of KBPAP. See FIG. 2 which shows aview of TRAP.

The active site with the two iron ions and the phosphate are shown atthe top. The beginning and end of the repression loop (RL) are alsomarked.

The beta sheets have seven strands each and are mostly parallel and thedi-iron active site is found at the C-terminal side of the sheets. SeeFIG. 3 which shows a secondary structure diagrams of the catalyticdomains of TRAP.

After cleavage of the 21 amino acid N-terminal signal peptide, thesequential structure begins with five residues leading up to the firststrand (β1) in the middle of sheet 1. This strand constitutes the firstelement in the conserved β-α-β-α-β motif. Asp 14 on β1 coordinate theFe3+ ion and Asp 52 on β2 coordinate both the Fe2+ and Fe3+ ions. Atyrosine side chain, Tyr 55, also coordinates the Fe3+ ion and isresponsible for the characteristic colour of this enzyme, due to acharge relay mechanism between the Fe3+ ion and the tyrosine side chainoxygen atom¹⁸. The delta oxygen of Asn 91 on β3 coordinates the Fe2+ion. The sequence then adopts a helix-turn-helix structure followed by afinal strand on this side of the sheet, before crossing over to sheet 2.The strand β4 does not have a corresponding element in KBPAP. Sheet 2begins with two strands, β5 and β6, connected by a short turn. β6 isfollowed immediately by a short helix, α5. The region between Ser 145and Val 161 is not visible in the electron density map. The densityresumes at Val 161 and leads to α6 and β7. At the C-terminal edge of β7His 186 is coordinating the Fe2+ ion. The following loop contains tworesidues, Glu 194 and His 195, that is located close to the active site.The corresponding pair in KBPAP contains two histidine residues. Thestructure continues with two helices, α7 and α8, interrupted by aproline residue and followed by β8. A short loop between β9 and β10contains the last metal interaction site, where Nε of His 221 and Nδ ofHis 223 coordinates the Fe2+ and Fe3+ ions respectively. Sheet 2continues with three antiparallel strands, β9-β11, before crossing overback to sheet 1. Three sequential and antiparallel strands constitutesthe C-terminal part of the structure. The last four residues, Arg 303-Pro 306 cannot be observed.

There is one disulfide bridge visible in the structure, between Cys142on α5 and Cys 200 on α7. The two cysteines are located on differentsides of the cleavage site and agrees with SDS-polyacrylamide gelelectrophoresis studies with and without reducing agent, which suggestedthat the two substructures were connected by a disulfide[6].

There are two N-glycosylation motifs in the TRAP sequence. At theextension of the Asn 97 side chain extra density can be seen for onecarbohydrate moiety, which has been included in the model. For Asn 128no such density can be seen. The exposed nature and high B-factors ofthat side chain suggests that high mobility may be the cause for notobserving glycosylation at this site. See FIG. 4 showing a view ofa-carbons in TRAP. The active site and an N-glycosylation site at Asn 97are displayed.

The Active Site

The side chains coordinating the iron ions in TRAP all have theirequivalent in KBPAP as has been predicted[32]. See FIG. 5, a view of theactive site of TRAP.

When all atoms in these residues, two aspartic acids, one asparagine,three histidines and a tyrosine, are aligned, the r.m.s distance is 0.7Å. To achieve such similiarty in structure for the active site, the twoproteins also share a conserved overall structure. A large density isvisible near the two metals that agrees with the size of a phosphateion. The resolution of the data does not inform us of the orientation ofthis molecule. Likewise, no solvent molecules in the active site can bemodelled accurately based on the present data.

The protein surface in and around the active site can be used forstructure based drug design. This surface is created by atoms from thetwo metal ions and from the following amino acid residues: Asp14, Asp52,Tyr55, Phe56, Asn91, His92, His186, Tyr187, Glu194, His195His221,His223, Phe244, Asp246. These amino acids are conserved between the ratenzyme, for which we have determined the structure, and the humanenzyme. Their structure in the active site is therefore relevant for thedesign of modulators or inhibitors of the human TRAP.

The missing density for residues between Ser145 and Val161 raises thequestion of proteolytic activation. SDS-polyacrylamide gelelectrophoresis indicated that crystallized TRAP had been cleaved intotwo fragments. FIG. 6 shows a SDS-PAGE gel showing the contents of thecrystal containing drops. The first three lanes after marker were rununder reducing conditions and the last three in non-reducing conditions.The gel shows that the protein has been cleaved between the residuesforming the disulfide and where the sizes of the cleavage productscorresponds to a cleavage of the protein in the repressions loop.

These fragments corresponded well in size to a cleavage of the proteinin this region. We have in U.S. patent application Ser. No. 09/442,816shown that rat TRAP can be cleaved in this part of the sequence with aresulting increase in activity (41). We cannot at this stage determinewhether there is a question of a single nicking of the enzyme or if apart of the sequence has been excised. A single cleavage site would giverise to two loose ends that could be too mobile to be detected in theelectron density maps. We have labeled the region between α5 and α6 therepression loop, since its cleavage or removal increases the activity ofthe enzyme up to ten fold. Ser145 is in close proximity to the activesite and it is plausible that the uncleaved loop partly covers it andthereby interferes with activity. Interestingly, this loop domain ismissing in KBPAP.

Catalytic Mechanism

The similarity of TRAP with the catalytic domain of KBPAP suggests asimilar reaction mechanism for the two enzymes. Klabunde and coworkershave suggested a mechanism for hydrolysis of phosphate substrates byKBPAP, based on the crystal structures of this enzyme in unliganded formas well as in complex with the reaction product phosphate and with theinhibitor tungstate [31]. They conclude that the phosphate substrateinteracts with the Zn2+ion followed by a nucleophilic attack on thephosphorous by an hydroxide ion coordinated by the Fe3+ ion. Thenegatively charged transition state is believed to be stabilized by theZn2+ ion and by the imidazoles of three histidine residues His202 ,His295 and His296, where the latter is also suggested as a potentialproton donor to the leaving alcohol group. These histidines in KBPAPhave their structural counterparts in His92, Glu194 and His195 in TRAP.The side chain of Glu194 points down into the protein and is not inclose contact with the phosphate group and probably has less of aneffect on catalysis than His295 has in KBPAP. His92 and His195 on theother hand superposes closely on their KBPAP counterparts. The largedifference between KBPAP and TRAP in the active site is the nature ofthe M2+ ion, where KBPAP has a zink ion and TRAP an iron ion. Since thecharge can alter between +2 and +3 for iron ions, TRAP is more sensitivethan KBPAP to redox agents [21, 25, 28].

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What is claimed is:
 1. A method for structure-based drug design for aspecific modulator, activator or inhibitor of TRAP (tartrate-resistantand purple acid phosphatase) activity, comprising generating a compoundstructure using a crystalline form of mammalian TRAP activated bycleavage prior to crystallization with a protease, wherein thecrystalline form of the mammalian TRAP is capable of being used forX-ray studies, and wherein the crystalline form of the mammalian TRAPhas a crystal structure with atomic structural coordinates as given inTable 2, or with coordinates having a root mean square deviationtherefrom, with respect to conserved backbone atoms of the listed aminoacid sequence, of not more than 1.5 Å.
 2. The method according to claim1, wherein the crystalline form of activated TRAP is human or rat TRAP.3. The method according to claim 1, wherein the crystalline form ofactivated TRAP comprises heavy metals.
 4. The method according to claim3, wherein the heavy metals are Fe.
 5. The method according to claim 1,wherein the crystalline TRAP has a conserved surface with a crystalstructure which is created by atoms from two metal ions and from thefollowing amino acid residues: Asp14, Asp52, Tyr55, Phe56, Asn91, His92,His186, Tyr187, Glu194, His195, His221, His223, Phe244, and Asp246. 6.The method according to claim 1, wherein the crystalline form ofactivated TRAP is prepared in the presence of inorganic phosphate. 7.The method according to claim 1, wherein the crystalline form ofactivated TRAP is grown using a reservoir containing 0.1 M KH2PO4. 8.The method according to claim 1, wherein the crystalline form ofactivated TRAP has a structure comprising a double beta sheet sandwichsurrounded on both sides by alpha helices.
 9. The method according toclaim 8, wherein the double beta sheet sandwich has seven strands foreach beta sheet.
 10. The method according to claim 1, wherein thecrystalline form of activated TRAP has a disulfide bridge between Cys142and Cys200.
 11. The method according to claim 1, wherein the crystallineform of activated TRAP comprises Fe2+ and Fe3+, wherein after cleavageof a 21 amino acid N-terminal signal peptide the mammalian TRAPcomprises amino acid residues Tyr55, Asn91, His186, Asp14, Asp52, His221and His223, and wherein Tyr55, Asn91 and His186 coordinate the Fe2+,Asp14 coordinates the Fe3+, and Asp52, His221 and His223 coordinate boththe Fe2+ and the Fe3+.
 12. The method according to claim 1, wherein thecrystalline form of activated TRAP is prepared in the presence of salt.13. The method of claim 1, further comprising (a) generating a conservedsurface of the crystalline form of activated TRAP on a computer screen;(b) generating compounds having a spatial structure; and (c) determiningif the compounds from step (b) fit the conserved surface.
 14. The methodaccording to claim 13, wherein the crystalline form of activated TRAP ishuman or rat TRAP.
 15. The method according to claim 13, wherein thecrystalline form of activated TRAP comprises heavy metals.
 16. Themethod according to claim 15, wherein the heavy metals are Fe.
 17. Themethod according to claim 13, wherein the crystalline TRAP has aconserved surface with a crystal structure which is created by atomsfrom two metal ions and from the following amino acid residues: Asp14,Asp52, Tyr55, Phe56, Asn91, His92, His186, Tyr187, Glu194, His 195,His221, His223, Phe244, and Asp246.
 18. The method according to claim13, wherein the crystalline form of activated TRAP is prepared in thepresence of inorganic phosphate.
 19. The method according to claim 13,wherein the crystalline form of activated TRAP is grown using areservoir containing 0.1 M KH2PO4.
 20. The method according to claim 13,wherein the crystalline form of activated TRAP has a structurecomprising a double beta sheet sandwich surrounded on both sides byalpha helices.
 21. The method according to claim 20, wherein the doublebeta sleet sandwich has seven strands for each beta sheet.
 22. Themethod according to claim 13, wherein the crystalline form of activatedTRAP has a disulfide bridge between Cys142 and Cys200.
 23. The methodaccording to claim 13, wherein the crystalline form of activated TRAPcomprises Fe2+ and Fe3+, wherein after cleavage of a 21 amino acidN-terminal signal peptide the mammalian TRAP comprises amino acidresidues Tyr55, Asn91, His186, Asp14, Asp52, His221 and His223, andwherein Tyr55, Asn91 and His186 coordinate the Fe2+, Asp14 coordinatesthe Fe3+, and Asp52, His221 and His223 coordinate both the Fe2+ and theFe3+.
 24. The method according to claim 13, wherein the crystalline formof activated TRAP is prepared in the presence of salt.